CRYOPRESERVATION OF SUGARCANE EMBRYOGENIC CALLUS USING A SIMPLIFIED FREEZING PROCESS

A simplified freezing process was developed and successfully applied t o embryogenic calluses of three sugarcane commercial hybrids (Saccharu m sp. cv. CP 5243, C 91-301 and C 1051-73). To obtain optimal survival , the calluses were pretreated with a cryoprotective solution containi ng 10% DMSO and 0.3 to 0.75M sucrose. For freezing, the samples were i mmersed in an alcohol bath placed in a -40 degrees C freezer, thus all owing a freezing rate comprised between 0.4 and 0.6 degrees C/min. Sam ples were held at that temperature for 2 h before immersion in liquid nitrogen. The highest survival rates of cryopreserved calluses ranged between 20 and 94% depending on the variety, and fully developed plant lets could be obtained from regenerating calluses. Embryogenic calluse s of one variety were stored for 14 months in liquid nitrogen without any effect on their survival rate and plantlet production.